BMC Genomics
BioMed Central
Open Access
Research article
Construction and sequence sampling of deep-coverage, large-insert
BAC libraries for three model lepidopteran species
Chengcang Wu1,4, Dina Proestou2, Dorothy Carter2, Erica Nicholson2,
Filippe Santos1, Shaying Zhao3, Hong-Bin Zhang*1 and
Marian R Goldsmith*2
Address: 1Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843-2474, USA, 2Department of Biological Sciences,
University of Rhode Island, Kingston, RI 02881-0816, USA, 3The Institute for Genomic Research, 9712 Medical Center Dr, Rockville, MD 20850,
USA and 4Current address: Lucigen Corporation, 2120 West Greenview Dr, Middleton, WI 53562, USA
Email: Chengcang Wu - cwu@lucigen.com; Dina Proestou - dproestou@mail.uri.edu; Dorothy Carter - RosyCarter@gmail.com;
Erica Nicholson - ericanic@verizon.net; Filippe Santos - fsantos@tamu.edu; Shaying Zhao - szhao@bmb.uga.edu; HongBin Zhang* - hbz7049@tamu.edu; Marian R Goldsmith* - mki101@uri.edu
* Corresponding authors
Published: 26 June 2009
BMC Genomics 2009, 10:283
doi:10.1186/1471-2164-10-283
Received: 10 September 2008
Accepted: 26 June 2009
This article is available from: http://www.biomedcentral.com/1471-2164/10/283
© 2009 Wu et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect
model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these
insects are critical resources for many molecular studies, including physical mapping and genome
sequencing, but not available to date.
Results: We report the construction and characterization of six large-insert BAC libraries for the three
species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two
restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from
152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two
combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome
coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative
single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC
ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the
genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the
category of low complexity repeats, and are more abundant in retro-elements than DNA transposons.
Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple
repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested
that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most
conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved
significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences.
Conclusion: The high-quality and large-insert BAC libraries of the insects, together with the identified
BACs containing genes of interest, provide valuable information, resources and tools for comprehensive
understanding and studies of the insect genomes and for addressing many fundamental questions in
Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and
evolution of the insect genomes.
Page 1 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
Background
Large-insert bacterial artificial chromosome (BAC) libraries have been shown to be critical resources for many
aspects of molecular and genomic studies [1,2], such as
the positional cloning of genes [3] and quantitative trait
loci [4], comparative studies of synteny and gene organization among different species [5], as well as for local or
whole genome physical and genetic mapping and
sequencing [6-11]. Arrayed, large-insert DNA libraries
have provided the opportunity for researchers to analyze
and share information and resources on specific clones
[1,2,12,13]. Hundreds of BAC libraries have been constructed for microbe, plant and animal species
[1,2,6,7,12,13]. However, only a few large-insert BAC
libraries are available to date for insect species, especially
lepidopteran insects [10,11,14-17]. This could slow
progress for the comprehensive molecular and genomics
research of these clades.
Moths and butterflies, members of the insect order Lepidoptera, are the second most diverse group of animals,
with at least 150,000 named species [18]. They are widespread members of the ecosystem, playing important
roles as pollinators and prey, and are among the most
destructive agricultural pests. Clearly, Lepidoptera are
under-represented in terms of genomic resources and
knowledge relative to their biological and economic status. This research was designed mainly to construct comprehensive BAC library resources for two species of moths,
the tobacco hornworm, Manduca sexta and the tobacco
budworm, Heliothis virescens, and one species of butterfly,
the Müllerian mimic, Heliconius erato. These species have
genome sizes ranging from 400 to 500 Mb/haploid
genome (395 Mb for H. erato [19], 404 Mb for H. virescens
[20], and 500 Mb for M. sexta [J. S. Johnston, pers. communication]) and are widely-used models for studying
fundamental problems in neurobiology [21], olfaction
[22], development [23], and immune responses [24] (M.
sexta]; host feeding preferences [25] and evolution of
insecticide resistance [26] and sexual communication systems [27] (H. virescens); and wing pattern mimicry [(H.
erato) [28]. Moths and butterflies are estimated to have
diverged from each other at least 50–60 million years ago
[18]. The sphingid, M. sexta, is a member of the same
superfamily, Bombycoidea, as the domesticated silkworm, Bombyx mori, the current genome model for Lepidoptera [8,9], and the noctuid, H. virescens, is related to
other pest noctuids currently being used for genomic studies including Spodoptera frugiperda [16,29] and Helicoverpa
armigera [30]. Here, we report the construction and characterization of six large-insert BAC libraries for these species and the first insight into the constitution and
evolution of their genomes. The libraries will enable a
large community of scientists to isolate and study the
genes controlling these processes, provide new tools for
http://www.biomedcentral.com/1471-2164/10/283
lepidopteran systematics, and serve as critical resources
for comparative genomic studies and genome sequencing
of this important group of organisms.
Results
Development of procedures for preparation of highmolecular-weight (HMW) DNA
One of the most important steps toward construction of
high-quality BAC libraries is preparation of high-quality
megabase DNA. Since no procedure was available for
preparation of HMW DNA from these insects, we first
developed a method for megabase DNA preparation by
testing different DNA isolation buffer systems and tissues
collected at different developmental stages of the insects.
The results showed that the day-10 pupae (males and
females) of M. sexta and day-4 pupae (males and females)
of H. virescens and H. erato were most suitable for megabase DNA isolation using a buffer system containing 0.1
M NaCl, 10 mM Tris-HCl, 10 mM EDTA, pH 9.4, and
0.15% β-mercaptoethanol. The DNA isolated with this
method was not only large in size (> 1000 kb), but also
readily digestible and clonable, thus being well-suited for
BAC library construction.
Library construction
The major goal of this study was to develop BAC resources
that are widely usable for molecular and genomic studies
of the insects, including whole genome physical mapping
and sequencing. Therefore, we constructed two BAC
libraries for each species with BamHI and EcoRI in the BAC
vector pECBAC1. Table 1 summarizes the characteristics
of the six BAC libraries constructed. The libraries were
named MSB and MSR for M. sexta (MS) and B or R for
BamHI or EcoRI, respectively, HVB and HVR for H. virescens, and HEB and HER for H. erato. The insert sizes of the
library clones were estimated based on a random sample
of 200–300 BACs from each library digested with NotI, a
relatively rare cutter in lepidopteran DNA, and fractionated on pulsed-field gels. A typical pulsed field gel pattern
for a set of random clones selected from the MSB BAC
library is shown in Figure 1. The average insert sizes of the
libraries ranged from 150–175 kb, and the proportion of
the insert-empty clones was <5%. Each library contained
from 19,200 to 21,504 clones which were arrayed into
384-well microtiter plates. Based on the number of clones
and average insert sizes of each library, we estimated that
the genome coverage of each library ranged from 6 ×–8 ×
genome equivalents, with the two combined libraries of
each species having a genome coverage of 13.0 ×–16.3 ×
(Table 1). Based on this and previous studies [1,2,6,7,13],
these BAC library resources should be well-suited for
many kinds of molecular and genomic research, including
whole genome physical mapping and sequencing.
Page 2 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
Table 1: Basic parameters of lepidopteran BAC libraries
Library name
Species
Cloning
enzyme
Mean insert
size (kb)
Rate of insertempty clones
No. of clones
arrayed
No. of plates
arrayed
Estimated
genome
coverage
Genome
coverage
of combined
libraries
HEB
HER
HVB
HVR
MSB
MSR
H. erato
H. erato
H. virescens
H. virescens
M. sexta
M. sexta
BamHI
EcoRI
BamHI
EcoRI
BamHI
EcoRI
175
153
171
150
152
165
<5%
<2%
<2%
1%
<1%
<1%
19,200
19,200
21,504
19,584
19,968
21,504
50
50
56
51
52
56
8.1X
7.3X
9.0X
7.3X
6.0X
7.0X
15.4X
BAC library screening
As an independent test of genome coverage, to demonstrate the utility of the libraries, and to isolate BACs containing genes of importance, the libraries were robotically
double-spotted onto Nylon membrane filters in 3 × 3 format and screened with gene sequence-specific probes that
are of interest for studies of the lepidopteran models.
These included genes involved in olfaction (MsOR1,
MsOR3, and HvHR16), nerve axon growth and guidance
(MsNos128, MsEph, MsFasII, and MsPlexA), hormone
action (MsE75, and HvPTTH), wing patterning (Hewg,
Heptc, and HeCi), Bt toxin action (HvAPN120 and Hvcad),
and ribosomal protein structure (HvRpS4, HeRpS5,
HeRpS9, HeRpL3, and HeRpL10), some of which have also
served as anchor loci for comparative linkage mapping
[5,31]. Tables 2, 3 and 4 summarize the library screening
results. Although the number of hits for individual probes
varied widely, which may reflect the uneven distribution
of the clones constructed with a single enzyme, the average number of hits in a library was close to the expected
BAC
(MSB)clones
Figure
phoresis
BAC
1 library
randomly
andselected
analyzedfrom
by pulsed-field
the M. sexta
gelBam
electroHI
BAC clones randomly selected from the M. sexta
Bam HI (MSB) BAC library and analyzed by pulsedfield gel electrophoresis. BAC DNA was isolated,
digested with NotI to release the insert DNA from the cloning vector, run on a 1% agarose gel and stained with ethidium
bromide. The average insert size of the clones was estimated
to be 150 kb. The common band (7.5 kb) appearing in all
BAC lanes is from the pEBAC1 cloning vector. Outer lanes
are Lambda ladder PFG marker (New England BioLabs,
USA).
16.3X
13.0X
genome coverage estimated by the library insert sizes and
the genome size (Table 1). The library genome coverage
estimated by hybridization was slightly higher than the
values expected by the BAC library insert sizes and the
genome DNA content for the libraries constructed for H.
virescens (16.7 hits per probe vs. 16.3 ×), and slightly
lower for the libraries from M. sexta (12.3 hits per probe
vs. 13.0 ×) and H. erato (13.9 hits per probe vs. 15.4 ×).
BAC end sequences (BESs)
To validate the libraries further, estimate levels of contamination by microbial or organellar sequences and obtain
some information about the constitution of the insect
genomes, one 96-well plate per library, thus two 96-well
plates per species, was sequenced from both ends of each
clone. A total of 246–299 BESs were successfully generated for each species (Table 5). The sequences had an average read length of 630 nucleotides, with an average of 560
Q20 nucleotides. Analysis of the BESs indicated that there
was no evidence of contamination with DNA from
organelles, the E. coli host, or other microbes that were
potentially carried by the DNA source insects. The
sequences are registered in the trace archives of GenBank
described in Methods [see Additional file 1].
The GC and repeat element contents of the insect
genomes
To provide a first insight into the constitution of the insect
genomes, we analyzed the BESs using the RepeatMasker
program, with an emphasis on the contents of GC and
repeat elements including retroelements, DNA transposons, simple repeats, and low complexity repeats (Table
5 [see Additional files 2, 3 and 4]). The GC contents of the
genomes ranged from 32.35% (H. erato) to 36.18% (H.
virescens), with the genome of the butterfly having 3.34%
less GC than those of the moths. A total of 117.702 kb of
BESs was obtained from the genome of H. erato. The
sequence contained a total of 231 repeats, which is equivalent to 10.01% of the genome. Two hundred six of these
repeats were categorized as low complexity (7.86% of the
genome). This number contrasted with those of H. virescens and M. sexta. H. virescens had a total of 198.779 kb
Page 3 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
Table 2: Number of positive clones on the Manduca sexta library filters.
Library
MSR 7.0X
MSB 6.0X
Expected 13.0X
Hits per library
Hits per library
Total hits
Probe source
A. Nighorn
A. Nighorn
H. Robertson
H. Robertson
P. Copenhaver
P. Copenhaver
L. Riddiford
Probe
Function
Eph
Nos 128
MsOR1
MsOR3
Fasciclin II
Plexin A
E75 Exon 4/5
eph-ephrin signaling
nitric oxide signal transduction
olfactory receptor
olfactory receptor
neuronal cell recognition
axon guidance
ecdysone receptor
8
6
3
11
12
10
8
4
3
4
11
3
-3
12
9
7
22
15
10
11
Ave. no. hits per probe
8.3
4.7
12.3
Probes were derived from bacterial plasmids.
BESs from which a total of 103 repeats (3.16% of the
genome) were identified, of which 79 were categorized as
low complexity (1.51% of the genome). Similarly, a total
of 180.031 kb of BESs generated from the M. sexta BACs
were found to contain a total of 125 repeats (3.34% of the
genome), of which 103 were categorized as low complexity (1.86% of the genome). Therefore, 3.16–10.01% of the
lepidopteran genomes comprised repeat elements, of
which a majority was categorized as low complexity
repeats. The overall percentage of repeat elements was
approximately 3-fold larger for the butterfly than for the
moth genomes; further, the percentage of the low complexity repeats was > 4-fold larger for the butterfly than for
the moth genomes. Among the low complexity repeats, 11
were longer than 100 bp (244 bp for the largest low complexity repeat), all of which were obtained from H. erato,
whereas all remaining low complexity repeats obtained
from M. sexta and H. virescens were shorter than 100 bp.
Retrotransposons, transposons and simple repeats were
also identified in the BESs, but altogether they comprised
<1% of the genomes. Nevertheless, the percentage of sim-
ple sequence repeats in the butterfly genome (0.66%) was
about 2-fold higher than those of the moth genomes
(0.39% and 0.27%). Moreover, a total of 15 retro-elements were identified in the BESs of all three species
whereas only 3 DNA transposons were identified, suggesting that retro-elements are generally more abundant than
DNA transposons in these genomes.
BLAST hits of the BESs
Using the discontiguous Megablast program to query the
database of all organisms available in GenBank, we
searched for matches to BESs of the three species by
BLASTn after masking with the RepeatMasker program
(Tables 6 and 7 [see Additional files 5, 6 and 7]). One
hundred eight (43.9%) of the 246 H. erato BESs had a
total of 1,364 hits to GenBank sequences, with an average
of 12.6 hits per BES (range 1–74 hits) at the default evalue of 3.0–3.5E–140. Of the 299 H. virescens BESs, 134
(44.8%) had a total of 1,128 hits, with each BES having an
average of 8.4 hits (range 1–69 hits). Of the 273 M. sexta
BESs, 127 (34.1%) had a total of 1,516 hits, with each BES
having an average of 11.9 hits (range 1–57 hits; Table 6).
Table 3: Number of positive clones on Heliothis virescens library filters.
Library
HVR 7.3X
HVB 9.0X
Expected 16.3X
Probe
Function
Hits per library
Hits per library
Total Hits
Probe source
K390
K386°
L086°
APN120°
Cadherin-650
Cadherin-1 kb
HR 16
PTTH
cap binding protein
ribosomal protein S4
TIA-1
Midgut receptor
Bt receptor
Bt receptor
Olfactory receptor
peptide hormone
7
10
5
8
5.8
3
---
14.8
1.1
5.4
3
4.4
-17
18
22
11
10
11
10
3
17
18
R. Palli
R. Palli
R. Palli
S. Gill
L. Gahan
S. Gill
F. Gould
PCR/GenBank
Ave. no. hits per probe
7.6
9.1
16.7
Probes were derived from bacterial plasmids except for PTTH. Decimals in hits reflect extrapolation for filters that were not screened.
Page 4 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
Table 4: Number of positive clones on Heliconius erato library filters.
Library
HER 7.3X
HEB 8.1X
Expected 15.4X
Hits per library
Hits per library
Total hits
Probe source
O. McMillan
O. McMillan
O. McMillan
O. McMillan
O. McMillan
PCR/Genbank
Probe
Function
RpS5
RpS9
RpL3
RpL10
Wingless
Ef1-α
ribosomal protein S5
ribosomal protein S9
ribosomal protein L3
ribosomal protein L10
Signal transduction
Translation elongation factor
2
4
10
1
13
13
5
5
1
3
12
8
7
9
11
4
25
21
Ave. no. hits per probe
7.2
5.7
13.9
Probes were derived from PCR products amplified from genomic DNA.
We further examined the distribution of the BLASTn hits
of the BESs among different species (Tables 6 and 7 [see
Additional files 5, 6 and 7]). Of the 1,364 H. erato hits,
1,226 (89.9%) were from 38 insect species. The majority
of the latter hits were from other Heliconius species,
including H. melpomene, H. doris, H. himera, H. erato, and
H. cydno, whereas the remaining hits were from H. virescens, Helicoverpa armigera (cotton bollworm), and H. zea
(corn earworm). Exclusively, 1,059 (77.6%) of the 1,364
H. erato hits belonged to H. melpomene. Moreover, we
found that both ends of three H. erato BACs, LQCBT56
(HER08E08), LQCBU02 (HEB02A01), and LQCBU16
(HEB04B01), had more than 10 discontiguous homologous sequences each to H. melpomene BACs registered in
GenBank. These results suggested that this set of BACs
may represent homologous regions. Complete sequencing of the three H. erato BACs will provide more information about the extent of microsynteny and evolution
between these two species.
In comparison, 859 (83.5%) of the 1,029 hits for H. virescens BESs were from 52 insect species (Table 7). The top
Table 5: Analysis of the BESs of H. erato (HEB and HER), H. virescens (HVB and HVR), and M. sexta (MSB and MSR) using the
RepeatMasker program [47].
BAC library
Total BAC ends
sequenced
No. BAC ends with
sequences
Successful rate of BAC end
sequencing (%)
Average length Q20
sequences (bp)
Total length Q20
sequences (bp)
HEB/HER
HVB/HVR
MSB/MSR
384
384
384
246
299
273
64.06
77.86
71.09
478
665
659
117702
198779
180031
BAC library
GC content (%)
Length (bp)/no.
HEB/HER
HVB/HVR
MSB/MSR
32.35
36.18
35.19
11781/231
6287/103
6018/125
BAC library
All repeats
Rate in genome (%)
10.01
3.16
3.34
DNA transposons
Length (bp)/no.
Rate in genome (%)
HEB/HER
HVB/HVR
MSB/MSR
279/2
0
535/1
0.24
0
0.3
BAC library
Small RNA
Rate in genome (%)
Length (bp)/no.
HEB/HER
HVB/HVR
MSB/MSR
0
0.67
0.09
777/17
779/21
487/11
Retroelements
Length (bp)/no.
Rate in genome (%)
777/2
1178/3
1490/10
Rolling circles
Length (bp)/no.
Rate in genome (%)
701/1
0
0
Simple repeats
Rate in genome (%)
0.66
0.39
0.27
0.6
0
0
0.66
0.59
0.83
Small RNA
Length (bp)/no.
0
1331/2
166/1
Low complex repeats
Length (bp)/no.
Rate in genome (%)
9247/206
2999/79
3340/103
7.86
1.51
1.86
Page 5 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
four values for BES hits included sequences from B. mori,
H. melpomene, H. virescens, and M. sexta. Compared with
27 and 24 lepidopteran species with sequences hit by the
H. erato and M. sexta BESs, respectively, 41 lepidopteran
species were represented in sequences hit by the H. virescens BESs, even though similar numbers of BESs (246–
299) were queried for the three species. This suggested
that the H. virescens genome is a broader representative of
Lepidoptera than H. erato or M. sexta.
Discussion
We have constructed six BAC libraries for three lepidopteran model species (2 moths and 1 butterfly). These
libraries not only have large-insert sizes (150 – 175 kb)
and deep genome coverage (13 × – 17 ×), but also have a
low level of insert-empty clones (<5%) and no detected
contamination with DNA from organelles and microbes
potentially living on the source insects, as indicated by
BES analysis. Moreover, the genome coverage and quality
of the libraries have been verified independently by
screening high-density filters of the libraries with a set of
single-copy genes or ESTs. The observation that none of
the libraries was contaminated with microbial DNA
potentially carried by the source insects was expected,
because the self-contained non-feeding pupal stage used
as a DNA source for the library construction had purged
their guts at the end of larval development. However, we
did observe 6, 21 and 20 short sequences in the He, Hv
and Ms BESs, respectively, which were homologous to
viral, bacterial, and fungal sequences (Tables 6 and 7 [see
Additional files 5, 6 and 7]). We believe that the homologues are real, but not from sample contamination
because they sit in the middle of BESs. These results perhaps provide a line of preliminary evidence for the presence of microbial sequences in these lepidopteran
genomes, possibly by horizontal transfer. Similar findings
have been obtained in B. mori [35]. On the other hand,
considering the small fraction (~0.5%) of the BAC libraries sampled, a more direct test of organelle contamination
could be accomplished by using mitochondrial sequences
as probes for hybridization. Furthermore, since the libraries of each species were constructed with two restriction
enzymes (EcoRI and BamHI) complementary in the GC
content of their restriction sites, the genome coverage
should be much better distributed along the genome than
those constructed with a single enzyme [13,36]. Therefore, these libraries could provide useful resources for
comprehensive genomics research of the three model lepidopterans.
Of the 933 hits for M. sexta BESs, 530 (56.8%) were from
30 insect species, including 340 hits (36.4%) exclusive to
known sequences in M. sexta. Moreover, the highest numbers of M. sexta hits included sequences from vertebrates,
including mouse (90), human (81), and zebrafish (63),
and plants (50). By comparison to the genomic sequences
of H. erato and H. virescens, the M. sexta genome seems to
have evolved to be less unique as a lepidopteran, showing
more similarities to the genomic sequences of other animals and even plants.
It was also found that most of the homologous sequences
were shorter than 200 bp. However, large contiguous
homologous sequences (>200 bp) were found to be associated with lepidopteran genes encoding proteins
involved in hormone metabolism, structural proteins,
and metabolic enzymes [see Additional files 5, 6 and 7].
An independent search of the BESs using BLASTx in GenBank and ButterflyBase [32] yielded an average of 10.7
hits per species with high homology to confirmed coding
regions of identified genes at e-values less than 1E-10 and
bitscores in a range of 55–383 [see Additional file 8].
Additional hits were to ORFs with high similarity to features associated with retrotransposons, such as reverse
transcriptase, gag-pol polyprotein, and endonuclease [33]
and non-LTR transposons found in the silkworm genome,
such as TRAS and SART [34]. Due to the limited sequencing information, the BLASTn results and discovery of
putative genes are presented as potential features to be
confirmed by further analysis.
Table 6: BLASTn hits of the BESs of H. erato (HEB and HER), H. virescens (HVB and HVR), and M. sexta (MSB and MSR) against the
sequences of all organisms available in GenBank (June 2007) calculated by number of sequences.
HEB/HER
HVB/HVR
MSB/MSR
HEB/HER
HVB/HVR
MSB/MSR
Leps
Bm
Hm
He
Hv
Ms
Other insects
Animals
1174
793
459
23
264
41
1059
53
3
16
2
0
5
48
1
1
24
340
52
66
81
122
143
323
Human
Mouse
Zebrafish
Viruses/bacteria
Fungi
Plants
Insect total
Total
14
32
81
23
29
90
10
23
63
4
13
10
2
8
10
10
6
50
1226
859
530
1364
1029
933
Bm, Bombyx mori; He, Heliconius erato; Hm, H. melpomene; Hv, Heliothis virescens; and Ms, Manduca sexta.
Page 6 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
Table 7: BLASTn hits of the BESs of H. erato (HEB and HER), H. virescens (HVB and HVR), and M. sexta (MSB and MSR) against the
sequences of all organisms available in GenBank (June 2007) calculated by number of species.
HEB/HER
HVB/HVR
MSB/MSR
Leps
Other insects
Animals
Viruses/bacteria
Fungi
Plants
Total
27
41
24
11
11
6
28
22
28
3
9
9
3
6
7
4
3
7
76
92
81
The libraries, library filters and individual clones have
been distributed to a number of laboratories and are presently being used for following studies: 1) walking to wing
colour patterning genes from closely linked AFLP
sequences in H. erato [17,37]; 2) testing synteny between
M. sexta, B. mori, and H. melpomene by chromosomal fluorescence in situ hybridization using BACs containing
orthologous genes as probes [5,38]; 3) analysis of fulllength coding and regulatory regions for the M. sexta Broad
gene (L. Riddiford, personal communication); and 4)
analysis of H. virescens HR16 putative odor receptor
sequences (F. Gould, personal communication).
The results of this study (Tables 5, 6 and 7) have provided
a snapshot of the basic characters of the genomes of a
group of ditrysian moths and butterflies which diverged
from each other at least 50–60 million years ago [18].
First, the genomes of all three species are AT-rich (64–
68%), with the genome of the butterfly (H. erato) having
an AT content more than 3% higher than those of the
moths (M. sexta and H. virescens). Second, the results show
that all three insect genomes contain relatively small fractions of repeat elements (3–10%), including retro-transposons, transposons, simple repeats, and low complexity
repeats. These results are in agreement with the small
genomes of the species (400–500 Mb/1C) which generally tend to contain smaller fractions of repeat elements.
Of these three insect species, the butterfly genome contains 3–5-fold more repeat elements (10.01% all repeats),
especially low complexity repeats, than the two moth
genomes. Papa reported that the total repetitive sequences
accounted for about 26% of the genomic regions linked to
wing pattern variation in H. erato [37]. The difference
could be an effect of more H. erato-specific repeats documented, sampling of a specific region with a higher average repeat density, or both. Third, whereas the three insect
genomes all contain a small number (<1%) of retro-elements, DNA transposons and simple repeats, retro-elements seem much more abundant than DNA
transposons, and the butterfly genome is two-fold richer
in simple repeats than the two moth genomes. Compared
with published information from B. mori, the finding of
such a low percentage of repeat contents in these three
lepidopteran species is surprising, especially for M. sexta,
which is in the same superfamily as the silkworm, Bombycoidea. Xia et al. [9] estimated about 20% of the B. mori
genome to be composed of "transposable elements;" further, early work based on Cot hybridization kinetics estimated about 45% of the silkworm genome to be
composed of repetitive sequences [39]. More recently
Osanai-Futahashi et al. reported that the TEs made up
35% of the silkworm genome and contributed greatly to
the genome size [40]. One may argue that we might simply have not identified all the relevant repeats in the BESs,
but our argument is supported by the following evidence.
The genome of the butterfly, H. erato, contains extremely
large numbers (1059 of 1364 hits) of small duplicated
sequences or "novel repeats" (not registered in GenBank)
which are homologous to three completely sequenced
BAC clones (118 kb of AEHM-41C10, 112 kb of AEHM46M10, and 118 kb of AEHM-7G12) of H. melpomene.
This in turn indicates the presence of novel repetitive or
duplicated sequences in the H. melpomene genome [see
Additional file 5]. Large-scale end sequencing of the complete BAC libraries will uncover more detailed aspects of
these butterfly and moth genomes, and provide more
information for fundamental studies of lepidopteran
insects in general.
The BLAST analysis of the sampled BESs has also provided
insights into the evolution of these insect genomes. It is
not surprising to find the top hits are to the sequences of
lepidopteran species, but it is quite surprising that the
highest numbers of M. sexta BES hits were to the
sequences of other animals and plants rather than to B.
mori (Tables 6 and 7). This finding suggests that although
all the genomes have undergone changes since the split
from the most recent common ancestor, they may have
done so along different trajectories, with the M. sexta
genome retaining some sequences in common with
plants and animals that have been either lost or modified
to a greater extent in H. virescens and H. erato. Such a
hypothesis can only be tested when more genomic data
are available for these lepidopteran insects. Moreover, the
BESs of the butterfly (H. erato) are well-matched only to
the sequences of H. melpomene. This suggests that not only
is the butterfly more related to H. melpomene than to the
two moth species, as expected, but this group has also
diverged to a greater extent, resulting in a higher level of
species- or evolutionary lineage-specific sequences. This
argument is further supported by the finding that 27 of
the 76 species having sequence matches to the BESs of H.
Page 7 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
erato (35.5%) were from other Lepidoptera. This number
is 6% higher than that of M. sexta but 9% less than that of
H. virescens. By contrast, the total number of top hits to
lepidopteran species for H. virescens BESs was 41, or 17
and 14 more than for M. sexta and H. erato, respectively.
Therefore, the genome of H. virescens may be a better representative of the genomes of Lepidoptera as a whole
(Table 7).
One may argue that the RepeatMasker program might not
mask the repeat sequences completely because of the limited amount of repeat elements available in the public
database; however, this does not appear to have affected
the BLAST results significantly. For instance, B. mori represents the species having the most sequence information in
GenBank among the lepidopteran species; however, we
found significantly different hits, 23, 264 and 41 for the
BESs of H. erato, H. virescens, and M. sexta, respectively
(Table 6). Moreover, there are many more Drosophila spp.
sequences in GenBank than for any other insect; however,
we only observed limited numbers of Drosophila sequence
hits: 25, 16 and 36 for the BESs of H. erato, H. virescens,
and M. sexta, respectively (not shown); there were no large
(≥ 200 bp) hits for any Drosophila sequence, and only one
large (≥ 200 bp) hit each for Apis mellifera (honey bee), for
the BESs of H. virescens and M. sexta, even though the
honey bee genome is also fully sequenced [see Additional
files 5, 6 and 7]. Similarly, a large number of top BLASTx
hits were to protein sequences in lepidopteran species
(12/32) or other insects (17/32), such as Acyrthosiphon
pisum (pea aphid) and Tribolium castaneum (red flour beetle), of which relatively few were to Drosophila spp (3/32).
Conclusion
We constructed six high-quality, deep-genome coverage
BAC libraries, two libraries for each of three lepidopteran
model species: H. erato, H. virescens, and M. sexta, with two
restriction enzymes, respectively. As the average clone
insert size of the libraries ranging from 152–175 kb, we
estimated that the genome coverage of each library ranged
from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes.
This genome coverage should be sufficient for many
aspects, if not all, of genomics studies of each species,
including genome-wide physical mapping and genome
sequencing.
Genomic sequence sample analysis of the moths and butterfly has provided an initial insight into the constitution
and evolution of their genomes. Although large-scale
genome sequencing is needed to further decipher the
genomes of the species, especially their gene contents, the
basic characteristics of the repeated sequence portion of
each genome is useful information for our understanding
of the genomes and their evolution. The high-quality BAC
http://www.biomedcentral.com/1471-2164/10/283
libraries of the insects, together with the gene-containing
BACs and BAC end sequences, provide valuable information, resources and tools for comprehensive studies of the
insect genomes and for addressing many fundamental
questions in Lepidoptera.
Methods
Insect Materials
Source of DNA
To minimize the potential polymorphism of the source
DNA for BAC library construction, we sought insects that
were as inbred as possible. For each species, we used progeny from a single pair mating to restrict the potential polymorphism of the insects to a maximum of 4 alleles per
locus. Because the source strains were at least partially
inbred, we expected significantly less polymorphism at
many loci. This strategy also minimized the number of
haplotypes in a library, since intra-chromosomal
exchange (crossing over) occurs only in lepidopteran
males. The source of DNA for M. sexta was a colony that
was maintained without outcrossing for at least 30 years
(L. Riddiford, U. Washington). For H. virescens (F. Gould,
North Carolina State U.) and H. erato (O. McMillan, U.
Puerto Rico), the DNA source was a colony that had to be
replenished periodically from wild populations to avoid
inbreeding depression. Whereas M. sexta and H. virescens
deposit large numbers of eggs in a short time, enabling a
relatively synchronous rearing, H. erato lays only a few
eggs each day for several months. Therefore, we collected
and froze insects at an appropriate stage (day-4 pupae)
based on pilot studies. Consequently, it took more than 6
months to accumulate a sufficient number of animals
(~200) to prepare high molecular weight (HMW) DNA
for library construction of this species.
Vouchering
To maintain the identity of the DNA source animals,
voucher specimens from the same families used for BAC
library construction were archived at the Museum of
Comparative Zoology, Harvard University (M. sexta, N.
Pierce), and at the North Carolina State University Insect
Collection (H. virescens and H. erato, F. Gould). The
archived specimens included dried adult wings and the
corresponding bodies preserved in 70–100% alcohol at 20°C or -80°C.
BAC library construction
The pECBAC1 vector [41] was used in the library construction [42]. Vector DNA was isolated by the alkaline lysis
method, purified by cesium chloride gradient centrifugation, digested completely with either BamHI or EcoRI, and
dephosphorylated with calf intestinal alkaline phosphatase. The digested vector DNA was precipitated, dissolved in TE (10 mM TrisHCl, 1 mM EDTA, pH 8.0),
Page 8 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
adjusted to 10 ng/μl, and stored at -20°C before use
[1,2,13,43].
HMW DNA was isolated from the insects using frozen
pupal tissues and buffer system (see Results) according to
the procedure described by Wu et al. [14]. The BAC libraries were constructed using an improved procedure developed in our laboratory [1,2,13,43]. Briefly, HMW
genomic DNA plugs were prepared from day-10 pupae
(males and females) of M. sexta and day-4 pupae (males
and females) of H. virescens and H. erato. DNA was partially digested with BamHI or EcoRI, size-fractionated in a
clamped homogeneous electrical field (CHEF) apparatus
(Bio-Rad), recovered by electroelution, and then ligated
into the BamHI or EcoRI site of the pECBAC1 vector,
respectively. The ligated DNA was transformed into E. coli
DH10B cells (Invitrogen, USA) by electroporation. The
transformed cells were incubated in SOC medium with
shaking at 250 rpm, 37°C for 1 h. Recombinant transformants were selected and incubated for 32 h at 37°C on
LB agar (Invitrogen, USA) plates containing 12.5 μg/ml
chloramphenicol, 0.5 mM IPTG, and 50 μg/ml X-gal.
Insert size analysis and BAC library arraying
White colonies were randomly selected and grown in LB
medium (Invitrogen, USA). BAC DNA was isolated,
digested with NotI, and subjected to CHEF gel electrophoresis. The ligation that gave a transformation efficiency of 200 or more white colonies/μl ligation and that
generated clones with the largest inserts was selected for
library construction. The BAC colonies were manually
arrayed as individual clones in 384-well microtiter plates
containing 50 μl LB plus freezing broth with 12.5 μg/ml
chloramphenicol [1,2,13,43,44]. After incubation at
37°C for 14 h, the microtiter plates were stored at -80°C.
To facilitate their accessibility, all six BAC libraries have
been made available to the public at the TAMU GENE
finder Genomic Resources Center directed by H.-B. Z.
BAC library screening
A GeneTAC G3 robotic workstation (Genomic Solutions,
Inc., USA) was used to double-spot the BAC libraries onto
8 × 12-cm Hybond N+ filters (Amersham-Pharmacia,
USA) in 3 × 3 format so that each high-density clone filter
contained two spots of each clone from four 384-well
microtiter plates (1,536 × 2 spots). The filters were processed according to Zhang [43] and Zhang et al. [44]. Filter
screening was carried out using a non-radioactive detection system (ECL, Pharmacia Amersham/Pharmacia,
USA) with X-ray film (Hyperfilm; Amersham/Pharmacia,
USA) according to the manufacturer's instructions. All
probes used except for putative olfactory receptors
(MsOR1, MsOR3, and HvHR16) were verified as single
copy by BLASTn search of KAIKObase [45] or BLASTx
search of FlyBase [46] to confirm the presence of one
http://www.biomedcentral.com/1471-2164/10/283
chromosomal locus. The amount of hybridizing DNA per
filter was adjusted to a range of 30–60 ng per filter based
on the intensity of signal obtained after initial screening.
We routinely used 0.5 M NaCl in the hybridization buffer,
but in some cases increased stringency to 0.4 M to reduce
background. We re-used filters without stripping until the
background became too high to read positive signals reliably or until we detected carry-through; then we treated
the filters to remove the probe DNA according to the manufacturer's instructions. Probe DNA was obtained from a
variety of sources, including bacterial plasmids containing
well-characterized cDNA sequences and PCR fragments
amplified from genomic DNA based on sequences registered in GenBank. Insert DNA was amplified by PCR using
primers designed from the plasmid vectors or within the
insert sequence and purified on Wizard Spin columns
(Promega, USA) before labelling with the ECL reagents.
Although we were able to detect positive signals on filters
hybridized with probes as short as 350–400 bp, probes of
1 kb or greater gave more consistent signal-to-noise ratios.
BAC end sequencing and analysis
Ninety-six clones were randomly selected from each of the
six BAC libraries and re-arrayed into a 96-well plate. Both
ends of each clone were sequenced using the primer 5'TAATACGACTCACTATAGGG-3' for the T7 end and 5'GTTTTTTGCGATCTGCCGTTTC-3' for the SP6 end using
the procedure developed at The Institute for Genomic
Research [12] [see Additional file 1 for the original library
clone names and the corresponding TIGR names]. The
sequences are registered in the trace archives of GenBank
under the following link: http://www.ncbi.nlm.nih.gov/
Traces/trace.cgi? SPECIES_CODE='HELICONIUS ERATO'
AND CENTER_NAME='TIGR' or TI#: 908600791908601036; SPECIES_CODE='HELIOTHIS VIRESCENS'
AND CENTER_NAME='TIGR' or TI#: 908601037–
908601335; and SPECIES_CODE='MANDUCA SEXTA'
AND CENTER_NAME='TIGR' or TI#: 908601336–
908601608. The resultant BESs were analyzed by utilizing
the RepeatMasker program [47]. Finally, the masked BESs
were BLASTed against the databases of all organisms by
using the discontiguous megablast program at NCBI using
the default criteria.
BLASTx searches were carried out against non-redundant
protein sequences in GenBank (December 2008) and ButterflyBase version 2.92 [32]. Hits with e-values less than
1E-7 and bitscores greater than 50 were evaluated for similarity to coding regions of identified proteins and retrotransposons. High matches of similar sequences in more
than one species was used as a criterion for provisional
identification of a bona fide protein.
Page 9 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
http://www.biomedcentral.com/1471-2164/10/283
Abbreviations
Additional file 6
BAC: Bacterial Artificial Chromosome; BES: BAC end
sequence; ORF: open reading frame.
Heliothis virescens BES BLASTn hits. Table S6 lists the sequences hit
by BLASTn search with H. virescens BESs and their characteristics.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S6.xls]
Authors' contributions
CW participated in the study design, BAC library construction, BAC end sequence data analysis and manuscript
preparation. DP, DC and EN participated in the library
screening. FS supported the BAC library assembly and filter preparation. SZ conducted the BAC end sequencing. HBZ participated in the study design, BAC library construction and manuscript preparation. MRG participated in the
study design, insect vouchering, library screening, BLASTx
search and manuscript preparation. All authors read and
approved the final manuscript.
Additional file 7
Manduca sexta BES BLASTn hits. Table S7 lists the sequences hit by
BLASTn search with M. sexta BESs and their characteristics.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S7.xls]
Additional file 8
High scoring BES hits identified by BLASTx search. Table S8 lists the
high scoring BLASTx hits in ButterflyBase and GenBank for H. erato, H.
virescens, and M. sexta
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S8.xls]
Additional material
Additional file 1
Correspondence of TIGR BAC end sequence names with their library
clone names. Table S1 lists TIGR BAC end sequence names and corresponding BAC library clone names.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S1.doc]
Additional file 2
Classes of repeats in BESs of 3 lepidopteran species analyzed with the
RepeatMasker tool. Table S2 includes summaries of types of repeated
sequences identified in the BESs of H. erato, H. virescens, and M. sexta
using RepeatMasker.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S2.xls]
Additional file 3
Lists of masked Lepidoptera repeats. Table S3 lists the masked repeats in
the BESs of H. erato, H. virescens, and M. sexta, their sequence characteristics, and location in the BESs.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S3.xls]
Acknowledgements
We acknowledge L. Riddiford at University of Washington, F. Gould at
North Carolina State University, and O. McMillan at University of Puerto
Rico for generous gifts of insects; and A. Nighorn, Arizona State University,
H. Robertson, University of Illinois, L. Riddiford, X. Zhou, University of
Washington, P. Copenhaver, Oregon Health and Science University, O.
McMillan, S. Gill, University of California at Riverside, L. Gahan, Clemson
University, F. Gould, North Carolina State University, and R. Palli, University of Kentucky, for probes. We also would like to thank the BAC end
sequencing team at TIGR for BAC end sequencing, N. Pierce at Harvard
University and F. Gould for archiving the insect specimens, and K. Pennoyer, University of Rhode Island, for the BLASTx search. This work was
funded by NSF Grant IBN-0208388 to MRG, CW, and H-BZ.
References
1.
2.
Additional file 4
Detailed repeat element contents in the BESs of three lepidopteran
models. Table S4 summarizes the types of repeat elements found in the
BESs of H. erato, H. virescens, and M. sexta.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S4.doc]
3.
4.
Additional file 5
Heliconius erato BES BLASTn hits. Table S5 lists the sequences hit by
BLASTn search with H. erato BESs and their characteristics.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-283-S5.xls]
5.
6.
Wu C, Xu Z, Zhang H-B: DNA libraries. In Encyclopedia of Molecular
Cell Biology And Molecular Medicine Volume 3. 2nd edition. Edited by:
Meyers RA. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA;
2004:385-425.
He L, Du C, Li Y, Scheuring C, Zhang H-B: Large-insert bacterial
clone libraries and their applications. In Aquaculture Genome
Technologies Edited by: Liu Z. Ames, IA: Blackwell Publishing;
2008:68-90.
Ito K, Kidokoro K, Sezutsu H, Nohata J, Yamamoto K, Kobayashi I,
Uchino K, Kalyebi A, Eguchi R, Hara W, et al.: Deletion of a gene
encoding an amino acid transporter in the midgut membrane causes resistance to a Bombyx parvo-like virus. Proc
Natl Acad Sci USA 2008, 105:7523-7527.
Grisart B, Coppieters W, Farnir F, Karim L, Ford C, Berzi P, Cambisano N, Mni M, Reid S, Simon P, Spelman R, Georges M, Snell R:
Positional candidate cloning of a QTL in dairy cattle: identification of a missense mutation in the bovine DGAT1 gene
with major effect on milk yield and composition. Genome Res
2002, 12:222-231.
Sahara K, Yoshido A, Marec F, Fuková I, Zhang H-B, Wu C, Goldsmith
MR, Yasukochi Y: Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca
sexta revealed by five-color BAC-FISH.
Genome 2007,
50:1061-1065.
Zhang H-B, Wu C: BAC as tools for genome sequencing. Plant
Physiol Biochem 2001, 39:195-209.
Page 10 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Wu C, Sun S, Lee M-K, Xu ZY, Ren C, Zhang H-B: Whole genome
physical mapping: An overview on methods for DNA fingerprinting. In The Handbook Of Plant Genome Mapping: Genetic And
Physical Mapping Edited by: Meksem K, Kahl G. Weinheim: WileyVCH Verlag GmbH & Co. KGaA; 2005:257-284.
Mita K, Kasahara M, Sasaki S, Nagayasu Y, Yamada T, Kanamori H,
Namiki N, Kitagawa M, Yamashita H, Yasukochi Y, et al.: The
genome sequence of silkworm, Bombyx mori. DNA Res 2004,
11:27-35.
Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T,
Chai C, et al.: A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 2004, 306:1937-1940.
Yamamoto K, Narukawa J, Kadono-Okuda K, Nohata J, Sasanuma M,
Suetsugu Y, Banno Y, Fujii H, Goldsmith MR, Mita K: Construction
of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences. Genetics 2006, 173:151-161.
Yamamoto K, Nohata J, Kadono-Okuda K, Narukawa J, Sasanuma M,
Sasanuma SI, Minami H, Shimomura M, Suetsugu Y, Banno Y, Osoegawa K, de Jong PJ, Goldsmith MR, Mita K: A BAC-based integrated linkage map of the silkworm Bombyx mori. Genome Biol
2008, 9:R21.
Zhao S, Shatsman S, Ayodeji B, Geer K, Tsegaye G, Krol M, Gebregeorgis E, Shvartsbeyn A, Russell D, Overton L, Jiang L, Dimitrov G,
Tran K, Shetty J, Malek JA, Feldblyum T, Nierman WC, Fraser CM:
Mouse BAC ends quality assessment and sequence analyses.
Genome Res 2001, 11:1736-1745.
Ren C, Xu ZY, Sun S, Lee M-K, Wu C, Zhang H-B: Genomic libraries for physical mapping. In The Handbook Of Plant Genome Mapping: Genetic And Physical Mapping Edited by: Meksem K, Kahl G.
Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2005:173-213.
Wu C, Asakawa S, Shimizu N, Kawasaki S, Yasukochi Y: Construction and characterization of bacterial artificial chromosome
libraries from the silkworm, Bombyx mori. Mol Gen Genet 1999,
261:698-706.
Suetsugu Y, Minami H, Shimomura M, Sasanuma S, Narukawa J, Mita
K, Yamamoto K: End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries. BMC Genomics 2007, 8:314. doi:10.1186/1471-2164-8-314
d'Alençon E, Piffanelli P, Volkoff A-N, Sabau X, Gimenez S, Rocher J,
Cérutti P, Fournier P: A genomic BAC library and a new BACGFP vector to study the holocentric pest Spodoptera frugiperda. Insect Biochem Mol Biol 2004, 34:331-341.
Baxter SW, Papa R, Chamberlain N, Humphray SJ, Joron M, Morrison
C, Ffrench-Constant RH, McMillan WO, Jiggins CD: Convergent
evolution in the genetic basis of Müllerian mimicry in heliconius butterflies. Genetics 2008, 180:1567-1577.
Kristensen NP, Skalski AW: Phylogeny and palaeontology. In
Lepidoptera, Moths and Butterflies. 1. Evolution, Systematics, and Biogeography. Handbook of Zoologie, Part 35, Arthropoda: Insecta Volume 4.
Edited by: Kristensen NP. Berlin and New York: Walter de Gruyter;
1998:7-25.
Tobler A, Kapan D, Flanagan NS, Gonzalez C, Peterson E, Jiggins CD,
Johnston JS, Heckel DH, McMillan WO: First-generation linkage
map of the warningly colored butterfly Heliconius erato.
Heredity 2005, 94:408-417.
Taylor A, Zawadzki J, Black B, Kreitman M: Genome size and
endopolyploidy in pyrethroid-resistant and susceptible
strains of Heliothis virescens (Lepidoptera: Noctuidae). J Econ
Entomol 1993, 86:1030-34.
Cafferty P, Auld VJ: No pun intended: future directions in invertebrate glial cell migration studies. Neuron Glia Biol 2007,
3:45-54.
Wilson CH, Christensen TA, Nighorn AJ: Inhibition of nitric oxide
and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, Manduca sexta. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2007, 193:715-28.
Truman JW, Riddiford LM: Morphostatic actions of juvenile hormone. Insect Biochem Mol Biol 2007, 37:761-70.
Kanost MR, Nardi JB: Innate immune responses of Manduca
sexta. In Molecular Biology and Genetics of the Lepidoptera (Contemporary Topics in Entomology) Edited by: Goldsmith MR, Marec F. Boca
Raton: CRC Press; 2009:271-291.
Oppenheim SJ, Hopper KR: Genetics of host range in Lepidoptera. In Molecular Biology and Genetics of the Lepidoptera (Contemporary
http://www.biomedcentral.com/1471-2164/10/283
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
Topics in Entomology) Edited by: Goldsmith MR, Marec F. Boca Raton:
CRC Press; 2009:195-217.
Heckel DG, Gahan LJ, Baxter SW, Zhao JZ, Shelton AM, Gould F,
Tabashnik BE: The diversity of Bt resistance genes in species of
Lepidoptera. J Invertebr Pathol 2007, 95:192-7.
Sheck AL, Groot AT, Ward CM, Gemeno C, Wang J, Brownie C,
Schal C, Gould F: Genetics of sex pheromone blend differences
between Heliothis virescens and Heliothis subflexa: a chromosome mapping approach. J Evol Biol 2006, 19:600-17.
Joron M, Jiggins CD, Papanicolaou A, McMillan WO: Heliconius
wing patterns: an evo-devo model for understanding phenotypic diversity. Heredity 2006, 97:157-167.
Negre V, Hotelier T, Volkoff AN, Gimenez S, Cousserans F, Mita K,
Sabau X, Rocher J, Lopez-Ferber M, d'Alencon E, Audant P, Sabourault C, Bidegainberry V, Hilliou F, Fournier P: SPODOBASE: an
EST database for the lepidopteran crop pest Spodoptera.
BMC Bioinformatics 2006, 7:322.
Gordon K, Tay WT, Collinge D, Williams A, Batterham P: Genetics
and molecular biology of the major crop pest genus Helicoverpa. In Molecular Biology and Genetics of the Lepidoptera (Current Topics in Entomology) Edited by: Goldsmith MR, Marec F. Boca Raton FL:
CRC Press; 2009:219-238.
Pringle EG, Baxter SW, Webster CL, Papanicolaou A, Lee SF, Jiggins
CD: Synteny and chromosome evolution in the lepidoptera:
evidence from mapping in Heliconius melpomene. Genetics
2007, 177:417-426.
ButterflyBase The Gene Database for Butterflies and Moths
[http://butterflybase.ice.mpg.de/]
Beauregard A, Curcio MJ, Belfort M: The take and give between
retrotransposable elements and their hosts. Annu Rev Genet
2008, 42:587-617.
Fujiwara H, Osanai M, Matsumoto T, Kojima KK: Telomere-specific non-LTR retrotransposons and telomere maintenance
in the silkworm, Bombyx mori.
Chromosome Res 2005,
13:455-467.
Daimon T, Hamada K, Mita K, Okano K, Suzuki MG, Kobayashi M,
Shimada T: A Bombyx mori gene, BmChi-h, encodes a protein
homologous to bacterial and baculovirus chitinases. Insect
Biochem Mol Biol 2003, 33:749-759.
Wu C, Nimmakayala P, Santos F, Springman R, Meksem K, Lightfoot
D, Zhang H-B: Construction of a complementary BAC library
for soybean genome-wide physical mapping and whole
genome sequencing. Theor Appl Genet 2004, 109:1041-1050.
Papa R, Morrison CM, Walters JR, Counterman BA, Chen R, Halder
G, Ferguson L, Chamberlain N, Ffrench-Constant R, Kapan DD, Jiggins CD, Reed RD, McMillan WO: Highly conserved gene order
and numerous novel repetitive elements in genomic regions
linked to wing pattern variation in Heliconius butterflies.
BMC Genomics 2008, 9:345.
Yasukochi Y, Ashakumary LA, Baba K, Yoshido A, Sahara K: A second-generation integrated map of the silkworm reveals synteny and conserved gene order between lepidopteran
insects. Genetics 2006, 173:1319-1328.
Gage LP: The Bombyx mori genome: analysis by DNA reassociation kinetics. Chromosoma 1974, 45:27-42.
Osanai-Futahashi M, Suetsugu Y, Mita K, Fujiwara H: Genome-wide
screening and characterization of transposable elements
and their distribution analysis in the silkworm, Bombyx mori.
Insect Biochem Mol Biol 2008, 38:1046-1057.
Frijters ACJ, Zhang Z, van Damme M, Wang G-L, Ronald PC, Michelmore RW: Construction of a bacterial artificial chromosome
library containing large EcoR I and Hin dIII genomic fragments of lettuce. Theor Appl Genet 1997, 94:390-399.
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon
M: Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factorbased vector. Proc Natl Acad Sci USA 1992, 89:8794-8797.
Zhang H-B: Construction and manipulation of large-insert
bacterial clone libraries – manual. 2000 [http://hbz7.tamu.edu/
homelinks/tool/protocol.htm]. Texas A&M University, Texas
Zhang H-B, Choi S, Woo S-S, Li Z, Wing RA: Construction and
characterization of two rice bacterial artificial chromosome
libraries from the parents of a permanent recombinant
inbred mapping population. Mol Breed 1996, 2:11-24.
KAIKObase [http://sgp.dna.affrc.go.jp/KAIKObase/]
Page 11 of 12
(page number not for citation purposes)
BMC Genomics 2009, 10:283
46.
47.
http://www.biomedcentral.com/1471-2164/10/283
FlyBase: A database of Drosophila genes and genomes [http:/
/flybase.org/]
Smit AFA, Hubley R, Green P: RepeatMasker Open-3.0. 1996
[http://www.repeatmasker.org].
Publish with Bio Med Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
BioMedcentral
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
Page 12 of 12
(page number not for citation purposes)